In gene therapy for inherited metabolic disorder and other diseases, an introduced exogenous gene is expected to be continuously expressed over a long period of time. This objective has been achieved conventionally by incorporating genetic information into a chromosome of a host by means of a retrovirus vector. Safety is a concern, however, because there have been some clinical reports of cells turning cancerous under the influence of the integrated genes. A suggested approach to the problem is to develop a genetic information expression system which is able to exist stably and independently from chromosomes. The approach has been so far not successful.
Sendai virus is a negative-sense, single-stranded RNA virus of the paramyxovirus family. The virus is attracting attention for its characters as potential material for gene therapy vectors. The virus is not pathogenic for human beings. Transcription and replication of the virus take place inside the cytoplasm and does not affect genetic information of the host. Furthermore, the virus exhibits high gene expression activity and low species-specificity.
A process has been so far established which produces recombinant Sendai virus through transfection with an expression vector from which T7 RNA polymerase transcribes a complementary strand for the full length genome RNA of Sendai virus and an expression vector for each NP, P, and L gene related to the transcription and replication of Sendai virus in cultured cells in which T7 RNA polymerase is forcefully expressed using a vaccinia virus vector or a plasmid vector. This process is used to produce recombinant Sendai virus from a Sendai virus producing vector in which an exogenous gene is inserted. The process has been successfully applied to produce recombinant Sendai viruses from which one of the F, M, and HN Sendai virus genes is defected. Further applications are being studied to realize a protein production system by making these recombinant Sendai viruses express a desired protein.
Many research groups are exploring possible applications for gene therapy as a genetic information expression system that can exist independently from chromosomes by using recombinant Sendai viruses produced from these Sendai virus producing vectors. However, these Sendai virus vectors are based on the cytotoxic Z strain and have a gene defected from the viruses to reduce their cytotoxicity. The vector infected cells are killed in a single generation; safety is thus improved, but gene expression lasts no longer than a limited period.
In contrast, there are known strains of Sendai virus with various nature, one of which was reported in 1979 by Tetsuya YOSHIDA (currently, professor at the University of Hiroshima) and his colleagues. The strain (temperature-sensitive mutant strain C1.151) is sensitive to temperature, generating few virus particles at 38° C. At 32° C., replication cycle is activated, and the strain generates virus particles.
The inventors of the present invention have focused on the fact that Sendai virus temperature-sensitive mutant strain C1.151 generate few virus particles and causes persistent infection at 38° C. and cloned the full length genome cDNAs of strain C1.151 and its parent strain, or Nagoya strain, to assemble a Sendai virus vector from which a gene is expressed over a long period of time. The inventors have reconstituted a virus by combining various fragments cut out with restriction enzymes from full length gene (+) stranded cDNAs of the two strains, so as to investigate whether the combinations exhibit temperature sensitivity and persistent infectiveness. The investigation has discovered that persistent infectiveness requires more than one mutation among those mutations where, in the M gene and F gene of strain C1.151, amino acid residues at positions-69, -116, and -183 in an M protein form a glutamate (E), an alanine (A), and a serine (S) respectively and amino acid residues at positions-6, -115, and -137 in an F protein form an arginine (R), a leucine (L), and a threonine (T) respectively.
The inventors also inserted an exogenous gene expressing cassette into the full length genome cDNA to examine sustainability of resultant exogenous gene expression by recombinant Sendai virus. The expression in cultured cells was sustained for a short period due to the extinction of infected cells when the Sendai virus vector derived from the Z strain was used, whereas the expression was sustained for 4 months or even longer when the Sendai virus vector derived from strain C1.151 was used. Infecting a rat's colon, the Sendai virus vector derived from the Z strain sustained expression no longer than about 2 weeks. In contrast, the Sendai virus vector derived from strain C1.151 sustained expression at least 2 months in colon epithelial cells.
These findings have established the Sendai virus vector derived from strain C1.151 as a very useful vector capable of persistent expression of an introduced gene, including inside living bodies. Nevertheless, the mechanism of persistent infection is not well understood. In addition, it is hoped that the vector be modified, without losing its sustainability, so that it does not release infective particles and is non-transmissible, in order to improve safety of the vector.
Citation List
Patent Literature 1
WO97/16539
Patent Literature 2
WO00/70070
Patent Literature 3
Japanese Patent Application Publication, Tokukai, No. 2002-272465)
Patent Literature 4
Japanese Patent Application Publication, Tokukai, No. 2006-325531)
Patent Literature 5
Japanese Patent Application Publication, Tokukai, No. 2006-180780)
Non-Patent Literature 1
T. Yoshida et al. (1979) Virology 92, 139-154.